Steve Ealick's Research Group

Abstract:

Müller IB, Knöckel J, Groves MR, Jordanova R, Ealick SE, Walter RD, and Wrenger C. The Assembly of the Plasmodial PLP Synthase Complex Follows a Defined Course. PLoS ONE 3:e1815 (2008).

Background.

Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet The active form of vitamin B6 is pyridoxal 5'-phosphate (PLP). For its synthesis two enzymes, Pdxl and Pdx2, act together, forming a multimeric complex consisting of 12 Pdxl and 12 Pdx2 protomers.

Methodology/Principal Findings

Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, idenbfied by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdxl:Pdxl interface and play an important role in Pdxl complex assembly. Mutation of these residues to alanine impedes both Pdxl activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdxl, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK.residues results in a hexamedc assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdxl:Pdxl interface, it affects the assembly of hexameric Pdxl into a functional dodecamer, which is crucial for PLP synthesis.

Conclusions/Significance

Taken together, our data suggest that the assembly of a functional Pdxl:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.